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CASE
Detection of Lactoferrin in food by using HPLC method
Lactorferrin is widely used nutritional additives with rapid development in recent food industries. It commonly applies for antimicrobial properties,
antivirus affection, iron balance regulating and meditating marrow cells generation in human body etc. T
antivirus affection, iron balance regulating and meditating marrow cells generation in human body etc. T
Details
Experimental process：
1. Sample preparation
Liquid sample: Weighed 5g (Accurately to 0.01g) sample. (Contain lactoferrin 0.1mg - 10mg) in 50ml
centrifuge tube, prepared to be extracted.
Solid sample: Weighed 1g (Accurately to 0.01g) sample. (Contain lactoferrin 0,1mg - 10mg) in a 50ml
beaker, prepared to be extracted.
2. Extraction
Liquid sample: 5ml disodium hydrogen phosphate solution is added in to sample tube (marked as A),
oscillated with vortex generator 0.5min, centrifuged sample with 10000 r/min for 10mins under 4
degree. Transferred supernatant to 50ml centrifuge tube (marked as B) ; Added 5ml disodium
hydrogen phosphate into original tube A and repeated whole centrifuge process once again. Combined
both suspernatant into centrifuge tube B.
Solid sample: 5ml warm disodium hydrogen phosphate solution(less than 50 degree) is added into
beaker, stirred and mixed well after solid was completely dissolved. Transferred sample into 50ml
centrifuge tube (marked as C), added 5ml warm disodium hydrogen phosphate solution again into
original beaker and combined both solution. The sample should be oscillated with vortex generator
0.5min, then centrifuged under speed of 10000 r/min for 10mins under 4 degree. Take
supernatant in to new centrifuge tube (marked as D), added 5ml disodium hydrogen phosphate
solution in centrifuge tube C and repeated whole process again, combined both suspernatant into
centrifuge tube D.
3. Purification
The heparin affinity column was firstly added with 5 mL of disodium hydrogen phosphate solution.
After the treatment, the sample extract was added. After the sample solution completely flowed
out, it was rinsed with 10 mL of disodium hydrogen phosphate solution, and all the effluent was
discarded. Finally, elute with 2.5 mL of disodium hydrogen phosphate-sodium chloride solution,
collect all the effluent, and dilute to 2.5 mL with disodium hydrogen phosphate-sodium chloride
solution. The solution filtration membrane will be collected for liquid chromatography detection
and analysis.
4. Condition
Column: 8.462579.0001
CNW Athena C4,5 μm,300A,250 mm×4.6 mm
Detection: 280 nm
Column temperature: 30℃
Injection volume: 50 μL
Flow rate: 1.0 mL/min
Mobile phase: 0.1% Trifluoroacetic acid and Acetonitrile
1. Sample preparation
Liquid sample: Weighed 5g (Accurately to 0.01g) sample. (Contain lactoferrin 0.1mg - 10mg) in 50ml
centrifuge tube, prepared to be extracted.
Solid sample: Weighed 1g (Accurately to 0.01g) sample. (Contain lactoferrin 0,1mg - 10mg) in a 50ml
beaker, prepared to be extracted.
2. Extraction
Liquid sample: 5ml disodium hydrogen phosphate solution is added in to sample tube (marked as A),
oscillated with vortex generator 0.5min, centrifuged sample with 10000 r/min for 10mins under 4
degree. Transferred supernatant to 50ml centrifuge tube (marked as B) ; Added 5ml disodium
hydrogen phosphate into original tube A and repeated whole centrifuge process once again. Combined
both suspernatant into centrifuge tube B.
Solid sample: 5ml warm disodium hydrogen phosphate solution(less than 50 degree) is added into
beaker, stirred and mixed well after solid was completely dissolved. Transferred sample into 50ml
centrifuge tube (marked as C), added 5ml warm disodium hydrogen phosphate solution again into
original beaker and combined both solution. The sample should be oscillated with vortex generator
0.5min, then centrifuged under speed of 10000 r/min for 10mins under 4 degree. Take
supernatant in to new centrifuge tube (marked as D), added 5ml disodium hydrogen phosphate
solution in centrifuge tube C and repeated whole process again, combined both suspernatant into
centrifuge tube D.
3. Purification
The heparin affinity column was firstly added with 5 mL of disodium hydrogen phosphate solution.
After the treatment, the sample extract was added. After the sample solution completely flowed
out, it was rinsed with 10 mL of disodium hydrogen phosphate solution, and all the effluent was
discarded. Finally, elute with 2.5 mL of disodium hydrogen phosphate-sodium chloride solution,
collect all the effluent, and dilute to 2.5 mL with disodium hydrogen phosphate-sodium chloride
solution. The solution filtration membrane will be collected for liquid chromatography detection
and analysis.
4. Condition
Column: 8.462579.0001
CNW Athena C4,5 μm,300A,250 mm×4.6 mm
Detection: 280 nm
Column temperature: 30℃
Injection volume: 50 μL
Flow rate: 1.0 mL/min
Mobile phase: 0.1% Trifluoroacetic acid and Acetonitrile
